title: Evaluation of a real-time quantitative PCR to measure the wild Plasmodium falciparum infectivity rate in salivary glands of Anopheles gambiae.
abstract: BACKGROUND
  Evaluation of malaria sporozoite rates in the salivary glands of Anopheles gambiae is essential for estimating the number of infective mosquitoes, and consequently, the entomological inoculation rate (EIR). EIR is a key indicator for evaluating the risk of malaria transmission. Although the enzyme-linked immunosorbent assay specific for detecting the circumsporozoite protein (CSP-ELISA) is routinely used in the field, it presents several limitations. A multiplex PCR can also be used to detect the four species of Plasmodium in salivary glands. The aim of this study was to evaluate the efficacy of a real-time quantitative PCR in detecting and quantifying wild Plasmodium falciparum in the salivary glands of An. gambiae.
  
  
  METHODS
  Anopheles gambiae (n=364) were experimentally infected with blood from P. falciparum gametocyte carriers, and P. falciparum in the sporozoite stage were detected in salivary glands by using a real-time quantitative PCR (qPCR) assay. The sensitivity and specificity of this qPCR were compared with the multiplex PCR applied from the Padley method. CSP-ELISA was also performed on carcasses of the same mosquitoes.
  
  
  RESULTS
  The prevalence of P. falciparum and the intensity of infection were evaluated using qPCR. This method had a limit of detection of six sporozoites per μL based on standard curves. The number of P. falciparum genomes in the salivary gland samples reached 9,262 parasites/μL (mean: 254.5; 95% CI: 163.5-345.6). The qPCR showed a similar sensitivity (100%) and a high specificity (60%) compared to the multiplex PCR. The agreement between the two methods was "substantial" (κ = 0.63, P <0.05). The number of P. falciparum-positive mosquitoes evaluated with the qPCR (76%), multiplex PCR (59%), and CSP-ELISA (83%) was significantly different (P <0.005).
  
  
  CONCLUSIONS
  The qPCR assay can be used to detect P. falciparum in salivary glands of An. gambiae. The qPCR is highly sensitive and is more specific than multiplex PCR, allowing an accurate measure of infective An. gambiae. The results also showed that the CSP-ELISA overestimates the sporozoite rate, detecting sporozoites in the haemolymph in addition to the salivary glands.
authors.affiliation: Laboratoire MIVEGEC (UMR IRD 224 CNRS 5290 UM1-UM2), 911 Av, Agropolis, 34394 Montpellier Cedex 5, France. alexandra.marie@ird.fr
authors.forename: Alexandra
authors.initials: A
authors.lastname: Marie
authors.forename: Anne
authors.initials: A
authors.lastname: Boissière
authors.forename: Majoline Tchioffo
authors.initials: MT
authors.lastname: Tsapi
authors.forename: Anne
authors.initials: A
authors.lastname: Poinsignon
authors.forename: Parfait H
authors.initials: PH
authors.lastname: Awono-Ambéné
authors.forename: Isabelle
authors.initials: I
authors.lastname: Morlais
authors.forename: Franck
authors.initials: F
authors.lastname: Remoue
authors.forename: Sylvie
authors.initials: S
authors.lastname: Cornelie
mesh_terms: D000818:Animals; D000852:Anopheles / Q000469:parasitology*; D004772:Entomology / Q000379:methods*; D005260:Female; D059208:Parasite Load; D010274:Parasitology / Q000379:methods*; D010963:Plasmodium falciparum / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012469:Salivary Glands / Q000469:parasitology; D012680:Sensitivity and Specificity